**1. Introduction**

The protozoan parasite *Leishmania* (*Viannia*) *braziliensis* (henceforth: *L. braziliensis)* is the main causative agent of human tegumentary leishmaniasis in Latin America. Infection with *L. braziliensis* generally causes cutaneous lesions, with possible, severe, metastatic mucosal involvement, and it is difficult to cure with the first-line pentavalent antimonial drugs [1–4]. In spite of its importance, the biology of *L. braziliensis* has not been analysed extensively, in part due to the limited set of genetic manipulation tools developed or adapted to this species.

While Gene replacement using homologous recombination has proven a useful tool for testing gene function in Old World *Leishmania* spp. [5–7], yet—to our knowledge—no gene replacement analyses have been reported for *L. braziliensis*. However, a functional RNA interference (RNAi) machinery, predicted from the *L. braziliensis* genome sequence [8], was corroborated experimentally [9], allowing gene function analysis in this species [9,10]. The RNAi pathway and associated genes are absent in species of the *L*. (*Leishmania*) subgenus such as *L. major* and *L. donovani* [9]. However, RNAi-based gene knock-down is prone to off-target effects [11], which can confound phenotypic analyses.

Recently, the CRISPR (clustered regularly interspaced short palindromic repeats)–Cas9 (CRISPR-associated protein 9) technology is revolutionizing gene function studies in a wide range of organisms, due to its high efficiency, precision, relative simplicity, and versatility [12]. Using this tool, the Cas9 endonuclease can be directed to a specific genomic locus by a single guide RNA (sgRNA) to introduce a double-stranded break (DSB) in the target DNA [13]. DSBs compromise genomic integrity and are identified and repaired by the nuclear machinery by regulated and error-prone DNA repair pathways [14], and homologous donor DNA templates may be inserted introducing defined changes into the DNA near the DSB as part of the repair process [15].

CRISPR–Cas9-mediated gene targeting and gene editing (e.g., to generate point mutations, or add tags to endogenous genes) have been successfully developed and applied in kinetoplastids, including *Trypanosoma cruzi* [16], *T. brucei* [17,18], and several species of *Leishmania* [17,19–24], with the notable exception of New World *L*. (*Viannia*) species. This new technology has greatly improved the efficiency of gene targeting in *Leishmania* spp. over traditional homologous recombination-based gene replacement.

First, CRISPR–Cas9 allows the rapid generation of gene deletion or gene disruption mutants in the promastigote stage (within 1–2 weeks depending on the species); thus minimising the occurrence of compensatory adaptations in the parasites [21,25]. This is particularly the case when a gene required for optimal *in vitro* survival and/or growth is targeted [26], since *Leishmania* have the remarkable ability to adapt to environmental changes by chromosome copy number variations [27,28]. Second, the generation of CRISPR-derived null mutants is facilitated by the use of donor DNA repair cassettes (containing antibiotic selection markers) flanked by short homology arms targeting the gene of interest (GOI), in a single transfection [17,29]. Third, both single and multigene families can be targeted with this system [16,30], and it even allows simultaneous editing of multiple loci [24,30], as well as the identification of essential genes [20,30,31]. CRISPR gene editing also allows for *in situ* addition of flanking loxP sites to a gene of interest and the subsequent rapamycin-inducible gene deletion by dimerisable Cre (DiCre) recombinase [32,33]. This facilitates deletion of essential genes and observation of the cell biological and morphological effects on living cells in a time-dependent manner.

In the absence of a donor DNA repair template, *Leishmania* use microhomology-mediated end-joining (MMEJ) or single-strand annealing (SSA) to repair DSBs, both of which lead to deletions of various sizes that disrupt the targeted gene [20,30,31]. These DSB repair pathways (MMEJ and SSA) have a generally low efficiency in *Leishmania*, and SSA may result in unwanted deletions of adjacent genes [31]. Transfections of a donor DNA template to facilitate homology-directed repair significantly improves CRISPR–Cas9 gene targeting efficiency and specificity, and eases the identification of CRISPR-edited mutants in *Leishmania* [17,19,20,30,31].

In this study, we establish the CRISPR–Cas9 technology as an experimental tool for reverse genetics in *L. braziliensis* facilitating the generation of null mutants and the analysis of gene function in this important human pathogen. We applied a cloning-free, PCR-based CRISPR–Cas9 method that was used successfully in *Leishmania mexicana*, *L. major*, *L. donovani*, and *Trypanosoma brucei* for rapid and precise gene editing [17,21]. As a proof of principle, we first targeted an integrated transgene coding for enhanced green fluorescent protein (eGFP) and then replaced two single-copy genes of *L. braziliensis* encoding heat shock proteins HSP23 and HSP100. In addition, we show that functions of these genes are conserved in the *Viannia* subgenus of *Leishmania.*
